Central Dogma Lecture 15 RNA processing, splicing, and degradation Flashcards
(37 cards)
What processing of pre-mRNA needs to be done in bacteria?
RNAs are generally “ready to go”
How are RNAs transcribed in eukaryotes?
As precursors that need to be processed to yield final useful RNA
How do mRNAs need to be processed in humans?
-Capping
-Splicing
-Polyadenylation
-Export to cytoplasm
-Other RNAs are edited and bases are modified
What is the 5’ cap on mRNA
-A modification of the 1st mRNA nucleotide with 7-methylguanosine
-Added co-transcriptionally to mRNA
What is the purpose of the 5’ cap in mRNA?
-Protects the mRNA from nucleases
-Binds to specific complexes of proteins
-Recruits the ribosome for translation
How is the 5’ cap added?
-5’ cap is a 7-methylguanosine
-Initially the 5’ end has a triphosphate (pppNpNp…)
-Phosphohydrolase removes γ phosphate (ppNpNp…)
-Guanylytransferase uses GTP tp add a G (GpppNpNp…)
-Guanine-7-methyltransferase adds a methyl (m^7GpppNpNp)
-2’-O-methyltransferase adds another methyl (m^7GpppmNpNp…)
Where is the 5’ cap on mRNA
-7-methylguanosine is added “backwards” via an unusual 5’,5’-triphosphate linkage
-First two bases are also often methylated
-Not methylated in yeast
-Methylated in human cells
What is an intron?
-Named for intervening sequences
-Removed from mRNA
What is an “exon”?
-Named for expressed sequences
-Kept in mRNA
Introns in yeast
Only a minority of genes have introns
Introns in humans characteristics
-Most genes have introns, often genes average 8 introns/gene
-Introns are ~10x longer than exons in humans
-Exons usually <200 bp, introns can be 50-20,000 bp
-Genes can be 100,000 bases long and take hours to transcribe
-Average human gene is 8,t00 bp, 90% introns
What is the purpose of introns?
-Not yet known
-Probably not junk
-Possibly alternative splicing of genes provides diversity
-Allow for rapid protein evolution through domain addition/subtraction
Introns allow for alternative splicing to make isoforms
-Muscle protein α-tropomyosin gene has 7 isoforms
-Exon usage depends on presence of splicing factors in each tissue
Four classes of introns
-Group I
-Group II
-Eukaryotic mRNA introns
-tRNA introns
Group I and Group II introns
-Self-splicing
-First example of RNA catalysis
-Require no additional proteins or ATP
-In nuclear, mitochondrial, chloroplast, and phage genomes
Eukaryotic mRNA introns
-Spliced by catalytic RNAs (snRNPs) in spliceosomes
-The most common introns in humans
-Active site resembles Group II ribozyme
-Lots of ATP-dependent conformational switches
tRNA introns
-Spliced by protein enzymes
-Primary transcript cleaved by endonuclease
-Exons are joined by ATP-dependent ligase
What organism was used when RNA self-splicing was discovered?
-Used Tetrahymena thermophila
-Eukaryote easy to grow, has 20k copies of a particular gene that gets spliced
How was RNA self-splicing discovered
-mRNA to be spliced, MgCl2 and GTP, and nuclear extract (because they assumed they needed an enzyme) were added to a tube
-In gel electrophoresis for the negative control, they left out the nuclear extract and there was just as much splicing
-Eventually found that RNA can act as an enzyme, “ribozyme”
Self-splicing mechanism of group I introns
-Cell will splice two sequences together
-RNA folds into a shape that bods exogenous GTP (GMP or G also works)
-3’-OH of GTP attacks 5’ splice site
-GTP leaves and G at 3’ end of protein slips into a pocket
-Free 3’OH of the exon attacks the downstream splice site
-Releases the intron (modified with G) plus the ligated exons
Self-splicing of Group II introns
-Similar to group I introns, except:
-Nucleophile in the first step is the 2’-OH of internal A (not 3’ OH because A is aimed at the 5’ splice site by secondary structure, can’t just attack anywhere)
-Formation of a “lariat loop structure, in which the A has three phosphodiester bonds and one is a 2’,5’-phosphodiester linkage
How’s does the overview of spliceosomal intron splicing compare to Group II?
-Similar to Group II splicing
-Uses 2’-OH of an internal A within the intron as a nucleophile
-Forms a lariat intermediate
-The 3’-OH of newly revealed 3’ end attacks splice site
-Differs from Group II because exogenous proteins and RNAs are required
-Complex is called the spliceosome
What is the spliceosome and what is it made up of?
-A large complex that helps with mRNA splicing
-Made up of multiple specialized RNP complexes called small nuclear ribonucleoproteins (snRNPs) and dozens of other proteins
What are snRNAs?
-100-200 nucleotides long and make up snRNPs
-U1, U2, U4, U5, and U6 are abundant in the nuclei
